This is a competitive renewal application to further characterize the molecular and cellular facets of host immunity in high-risk corneal transplantation. High-risk corneal grafts are characterized by their rapid and high frequency of rejection. However, the precise molecular mechanisms that dictate their poor prognosis remain incompletely understood. The goal of these studies is to gain new insights into the mechanisms that distinguish high-risk vs. low-risk transplant immunity. We hypothesize that (i) induction of immunity to high- risk corneal transplants is characterized by a significantly enhanced capacity of alloantigen-bearing antigen- presenting cells (APC) to gain access to regional lymph nodes; (ii) allosensitization is enhanced in the high- risk transplant setting due to graft bed microenvironment changes that skew APC from a potentially tolerogenic phenotype to a mature highly sensitizing phenotype; and (iii) host regulatory T cells can be conditioned to maximize their homing and functional capacity for suppressing T cell responses so as to promote high-risk transplant survival. To test these hypotheses, we propose to pursue four specific aims: In Aim 1, we will investigate the precise and distinct contributions of corneal blood vessels and lymphatics in mediating cell trafficking, and the function of pro-lymphangiogenic mediators, VEGF-C, VEGF-D, and VEGFR-3, in regulating APC maturation and function. In Aim 2 we plan to enrich the donor graft with tolerogenic APC to counter the pro-maturation signals of the high-risk graft bed that promote sensitization. In Aim 3 we will evaluate the specific molecular and cellular regulation of APC homing to regional lymph nodes in transplantation. In Aim 4 we propose to amplify expression of lymph node homing receptors on T regulatory cells ex vivo to promote high-risk corneal transplant survival. Our study design relies on using the well-characterized mouse model of orthotopic corneal transplantation in conjunction with in vitro immunological assays, molecular and cell migration assays, and a variety of interventions including cytokine, chemokine, and endothelial growth factor ligand and receptor blockade strategies and bone marrow chimeras to gain mechanistic insights into the molecular regulation of corneal alloimmunity. The long-term objective is to use the information derived from these projects to develop new prophylactic and treatment strategies to promote high-risk graft acceptance. The overall health relevance of this research is that corneal grafting represents by far the number one form of tissue transplantation performed in the United States. However, most high-risk corneal transplant patients rapidly reject their grafts, there has been no significant change in the management or prognosis of high-risk transplantation in decades, and the immunosuppressive strategies currently in use are associated with significant toxicity and side-effects. PUBLIC HEALTH RELEVANCE: High-risk corneal transplantation, performed in inflamed host graft beds, is characterized by swift and often irreversible immune rejection. While tens of thousands of these are performed annually on a global basis, there has been no significant change in the prognosis of high-risk grafts for decades. This grant proposes to study the mechanisms that are involved in induction of immunity to high-risk corneal transplants and develop novel strategies to promote tolerance and long-term survival of these grafts without use of toxic immunosuppressive medications.